Virulence factors of Salmonella spp. isolated from free‐living grass snakes Natrix natrix

Abstract Salmonellosis associated with reptiles is a well‐researched topic, particularly in China and the United States, but it occurs less frequently in Europe. The growth of the human population and changes in the environment could potentially increase the interaction between humans and free‐living reptiles, which are an unidentified source of Salmonella species. In this study, we sought to explore this issue by comparing the microbiota of free‐living European grass snakes, scientifically known as Natrix natrix, with that of captive banded water snakes, or Nerodia fasciata. We were able to isolate 27 strains of Salmonella species from cloacal swabs of 59 N. natrix and 3 strains from 10 N. fasciata. Our findings revealed that free‐living snakes can carry strains of Salmonella species that are resistant to normal human serum (NHS). In contrast, all the Salmonella species strains isolated from N. fasciata were sensitive to the action of the NHS, further supporting our findings. We identified two serovars from N. natrix: Salmonella enterica subspecies diarizonae and S. enterica subspecies houtenae. Additionally, we identified three different virulotypes (VT) with invA, sipB, prgH, orgA, tolC, iroN, sitC, sifA, sopB, spiA, cdtB and msgA genes, and β‐galactosidase synthesised by 23 serovars. The identification of Salmonella species in terms of their VT is a relatively unknown aspect of their pathology. This can be specific to the serovar and pathovar and could be a result of adaptation to a new host or environment.


INTRODUCTION
Zoonoses constitute a major problem for public health.The World Health Organization (WHO) estimated that 60% of emerging infectious diseases have a zoonotic origin (WHO, 2021).Since January 2020, the world has struggled with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, which has a zoonotic origin (Holmes et al., 2021).This example also shows that zoonotic pathogens may lead to unexpected and barely controllable negative consequences for public health.Expanding knowledge about those pathogens, especially their virulence factors, is crucial for developing effective strategies to combat them.Therefore, even before the coronavirus disease 2019 (COVID-19) pandemic, the Centers for Disease Control and Prevention (CDC) created One Health, which is an approach focusing on zoonotic diseases as a global threat (CDC, 2018).One of the most widely known bacterial zoonotic pathogens, also pointed out by One Health, is Salmonella spp., a causative agent of foodborne diseases and gastrointestinal infections.In 2018, Salmonella spp. was the second most common causative agent of gastrointestinal infections in the European Union (EU), affecting 91,857 people (European Food Safety Authority [EFSA], 2019).Salmonella spp.rods transmitted to humans from animals are pathogenic to people-causing non-typhoidal salmonellosis (NTS).NTS is a global health problem, as annually worldwide, there are 1.3 billion cases of Salmonella spp.gastroenteritis, leading to 3 million deaths (Kurtz et al., 2017).There is still no vaccine for people, so examining transmission sources is essential to determine the ways of preventing salmonellosis.NTS is usually caused by oral transmission of Salmonella spp.by food and/or water but also by contact with animals.
Many animals can be vectors of salmonellosis, affecting people worldwide.For instance, in Europe, Salmonella spp.presence in the food chain mainly refers to eggs and poultry.However, a growing body of data indicates that one of the most essential vertebrates playing a role in spreading human salmonellosis is likely to be reptiles, known as asymptomatic carriers of Salmonella spp.rods, with a prevalence ranging up to 90% of reptile specimens.To date, many studies have identified the presence of more than one Salmonella spp.serovar in the reptile's gastrointestinal tracts (Corrente et al., 2004;Ebani et al., 2005;Geue & Löschner, 2002;Jang et al., 2008;Nakadai et al., 2005).Reptile-associated salmonellosis (RAS) occurs mainly in children under 5 years, elderly people, and immunocompromised patients.Approximately 15% of children's RAS cases are invasive (Zając et al., 2016).Salmonella spp.can penetrate and colonise their intestinal tract, leading to symptoms such as diarrhoea, abdominal cramps, fever, and vomiting.Several reports from the United States and other countries described RAS cases and their origin, identifying examples of both direct and indirect contact with reptiles (Baranzelli et al., 2017;Damborg et al., 2016;Gambino-Shirley et al., 2018;Gavrilovici et al., 2017;Horvath et al., 2016;Kiebler et al., 2020;Suzuki et al., 2017).
When analysing the National Library of Medicine's PubMed, after searching for 'Salmonella reptiles,' we found 239 results from the last 10 years (23 February 2024).Most of these studies come from the United States and China, while in Europe, the role of Salmonella spp.transmission connected with reptiles in the epidemiological chain is poorly examined (35 publications, PubMed 23 February 2024).In addition, the majority of these studies were conducted on captive animals maintained in zoological gardens, home collections, and households (Alix et al., 2008).There were only five original articles, including free-living reptiles in Europe (Krautwald-Junghanns et al., 2013;Nowakiewicz et al., 2015;Pawlak et al., 2020;Schmidt et al., 2014;Zając et al., 2016).
RAS is well known to cause numerous gastrointestinal infections in the United States, but in many European countries, this problem of public health goes unnoticed and is neglected.This disproportion shows the importance of such studies on reptiles free-living in Europe.As we still do not have sufficient reports about Salmonella spp.prevalence in the gastrointestinal tract of free-living European reptiles, the present study aims to fulfill this gap by scanning a large sample of freeliving snakes.We previously studied the Gramnegative aerobic microbiota among the free-living grass snake Natrix natrix (Pawlak et al., 2020).From a total of 45 studied reptiles, Salmonella species were isolated in 10 of them (10/45; 22.2%).In this study, we aim to expand the knowledge about previously and now collected Salmonella strains (27 strains collected from 59 N. natrix-snakes summed up from previous and current studies).Here, we aimed at detailed recognition of the virulence factors of all the collected strains with a wide array of approaches, including biochemical analysis, proteomic and serological identification, and analysis of the phylogenetic relationships of Salmonella spp.virulence genotyping, antimicrobial susceptibility, and human serum susceptibility.To show the impact of the habitat of reptiles on the composition of their intestinal microflora, we also compared 27 strains from the free-living N. natrix with 3 Salmonella spp.strains isolated from the gastrointestinal tracts of the captive-born banded water snake Nerodia fasciata.While N. fasciata and N. natrix are both semiaquatic snakes and share some ecological similarities, they are phylogenetically distinct, and the presence of N. fasciata in Europe is solely related to herpetoculture, as this species is native to North America (Hibbitts & Fitzgerald, 2005).Therefore, we put efforts into determining their similarities and differences in terms of virulence genotyping, antimicrobial and normal human serum (NHS) susceptibility profiles.

Sample collection from reptiles
We previously studied the Gram-negative aerobic microbiota of 45 free-living grass snakes N. natrix in Poland (Pawlak et al., 2020) determining that 22.2% of tested animals (n = 10) were carriers of Salmonella spp.Furthermore, we isolated Salmonella spp.from the other 14 free-living N. natrix in Poland, so the total number of snakes was 59 (n = 59), among which we detected 27 Salmonella spp.strains.The previous study was focused on only determining the species composition of the Gram-negative microbiota of freeliving snakes.In the present study, we focused on the detailed characteristics of Salmonella strains collected from free-living N. natrix.The samples were collected from the cloaca of free-living grass snakes (N.natrix-abbreviation: NN) in the Małopolskie Voivodeship (S Poland; vicinity of Krak ow and Niepolomice Forest; n = 59) in summer during the active season and also from banded water snake (N.fasciata-abbreviation: NF) obtained from a private breeding colony and then maintained in the laboratory (n = 10), as described before (Pawlak et al., 2020).Since the microbiota of snakes is likely to be strongly affected by absorptive state and recent diet, we transferred snakes to the laboratory in the Institute of Environmental Sciences of the Jagiellonian University in Krak ow to standardise sampling conditions.Snakes were maintained solitarily in disinfected terraria, with water provided ad libitum, and fed once per week.Before sampling, all snakes were fed with captive rodents to equalise the diet composition and get insight into the composition of Enterobacterales, which represent the core microbiota of snakes.Also, before being provided to snakes, rodents were kept at À20 C to deplete their own microbiota.Each snake was sampled at least 1 week after feeding to ensure it was in a post-absorptive state (Skoczylas, 1970).Samples were collected by placing the sterile swab inside the cloaca of the snakes for 30 s with gentle rotational movements.A unique identifier for each isolated Salmonella spp.strain was assigned during sample collection.Moreover, Salmonella spp.strains were deposited in the Polish Collection of Microorganisms, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

Identification of Salmonella spp. isolates
As environmental and clinical strains of some bacterial species, including Salmonella spp., present atypical biochemical features (Kontou et al., 2007;Mour ão et al., 2020;Mrozowska & Tyc, 1992), we decided to identify the bacterial strains using several identification methods.This ensures that the identification level is of the highest quality.

Colonial examination of Salmonella spp. isolates
For colonial examination, the bacteria were cultured overnight at 37 C in nutrient agar (Biocorp) for 24 h to obtain pure, rounded, non-pigmented and single colonies.This step was repeated three times.Following this incubation, a loop full of samples was streaked on MacConkey agar (Biocorp), Salmonella-Shigella (SS) agar (Biocorp), Simmons agar (Biocorp), xylose lysine deoxycholate (XLD) agar (Merck) and Chromogenic agar (Fluka Analytical) for 24 h at 37 C.All media (except SS and XLD agar) were autoclaved at 120 C for 15 min at 0.5 atm.We assessed the grown colonies based on visual observation by determining their morphological (shape, colour, size and surface appearance) and biochemical (growth in selective media) features.This analysis was validated by comparing the grown colonies with standard clinical Salmonella spp.samples described in the literature, acting, in our case, as a positive control (Salmonella enterica serovar Typhimurium ATCC 14028, S. enterica serovar Montevideo ATCC 8387 and S. enterica serovar Oranienburg ATCC 9239- Heithoff et al., 2008;Kim et al., 2023;Maddocks et al., 2002;Pao et al., 2005;Svanevik & Lunestad et al., 2015).At this point, all isolates were initially classified into typical or atypical strains based on the visual characterisation of each bacterial colony grown in the shown above media.

Biochemical identification of Salmonella spp. isolates
Biochemical identification of bacterial isolates was determined using the API 20E plate system purchased from Biomerieux, France (cat# 20-100).The experiment was performed and analysed by following the manufacturer's instructions.

Serological identification of Salmonella spp.
To determine the serological types of Salmonella spp.strains, the following antisera were used (Biomed, Poland): polyvalent HM antiserum (Lot No. 29420001) for Salmonella flagellar antigen, the different O group antisera: BO (Lot No. 25220001), CO (Lot No. 25321001), DO (Lot No. 25422001) and EO group (Lot No. 25520001) for the types of O-somatic antigen, as well as the H:g,m antiserum (Lot No. 27120001) for the flagellar factor characteristic of Salmonella Enteritidis.Colonies of each Salmonella spp.strain was suspended in a drop of physiological saline to identify the rough form (due to their ability to auto-agglutinate, which excludes them from the further course of the serotyping reaction).Strains showing negative autoagglutination results in physiological saline (the socalled smooth strains) were qualified for further study.Agglutination was performed in the polyvalent HM antiserum first, which allowed us to confirm the belonging of the strains to the genus Salmonella, and then with the O groups antisera and H:g,m flagellar factor serum.The agglutination ability was checked by suspending the bacteria in a drop of serum on a glass slide and then distributing the bacterial mass throughout the drop volume, obtaining a thick, milky suspension.The slides were slightly rocked for up to 60 s in each agglutination test.Visible cell aggregates were considered a positive result for agglutination ability.
Proteomic identification of Salmonella spp.isolates (matrix assisted laser desorption/ ionization -time of flight -mass spectrometry MALDI-TOF MS) Pure single colonies of actively growing bacterial cultures were suspended in 300 μL of distilled water, then mixed after the addition of 900 μL of absolute ethanol.The supernatant was aspirated, and the pellet was dried at room temperature.Then 50 μL of 70% formic acid and 50 μL of acetonitrile were added and mixed by pipetting, followed by centrifugation at 13,000Âg for 2 min.Next, 1 μL of supernatant was applied to the 384-spot ground steel target plate (Bruker Daltonics, Bremen, Germany) and air-dried at room temperature, followed by the addition of 1 μL of α-cyano-4-hydroxycinnamic acid matrix solution (HCCA; Bruker Daltonics) and air-dried again.Salmonella spp.strains were identified using a Bruker Daltonics UltrafleXtreme spectrometer under the control of flexControl software 3.4 (Bruker Daltonics).Biotyper 3.1 software (Bruker Daltonics) and a database containing 6904 entries were used for the identification.According to the manufacturer, the following score values were used: less than 1.7-identification not reliable, 1.7-2.0-probablegenus identification, 2.0-2.3 secure genus identification and probable species identification, and more than 2.3-highly probable species identification.

DNA extraction
DNA was extracted from Salmonella spp.isolates using a commercially available Genomic Mini Kit (A&A Biotechnology, Poland) according to the manufacturer's protocol from an overnight (18-24 h, 37 C) culture.
Analysis of genetic similarity between isolated Salmonella spp.strains

Virulence genotyping
Strains were subjected to the testing of 17 virulence genes (VG) related to the pathogenicity of Salmonella spp.The VG genes were targeted by three following multiplex-PCR reactions (I: invA, sipB, prgH, spaN, orgA and tolC genes; II: iroN, sitC, lpfC, sifA, sopB and pefA genes; and III: spvB, spiA, pagC, cdtB and msgA) performed according to Skyberg et al. (2006).The list of the primers used in this study (Genomed, Poland) and VGs functions is presented in Table 2.The amplification conditions for all three reactions were the same and set as follows: 95 C for 5 min of initial denaturation, 34 cycles of denaturation (40 s, 94 C), annealing (60 s, 66.5 C), extension (1.5 min, 72.5 C) and final extension (10 min, 72 C).PCR amplifications of each type of reaction were performed with a DNA Thermal Cycler T100 (Bio-Rad, USA).

Gel electrophoresis, visualisation and analysis of PCR amplification products
Amplicons were separated using a 2% agarose gel containing 4 μL of Midori Green (Nippon Genetics) at a constant voltage of 110 V for approximately 60 min using a Consort power supply.The mass marker ranged from 100 to 1000 bp (A&A Biotechnology, Cat # 3000-500).The amplified products were visualised with Midori Green DNA (Nippon Genetics, Germany) under UV light using a Gel Doc camera system (Bio-Rad, USA) and analysed with Quantity One software (Bio-Rad, USA).The dendrogram was generated using Bio-Numerics 7.6.2(Applied Maths) by Dice coefficient with optimisation of 0.5% and unweighted pair group method with arithmetic mean (UPGMA) clustering with a tolerance of 1.0%.The discriminatory power was calculated with Simpson's index of diversity (Hunter & Gaston, 1998).

Serum
To determine the sensitivity of Salmonella spp.strains isolated from snakes to bactericidal serum activity, we purchased the pooled blood human serum (NHS) from the Regional Center for Blood Donation and Blood Treatment (Wroclaw, Poland)

Normal human serum bactericidal activity
The bactericidal activity of 50% NHS was determined according to the assay as previously described (Pawlak et al., 2017).Strains isolated both from N. natrix (II 4.1S,39.1K,28.1K,NN 1.3 and NN 13.3) and N. fasciata (NF 9.2, NF 9.4 and NF 9.5) were selected for this test.Overnight culture (5 mL of Luria-Bertani [LB] medium) was transferred (150 μL) to fresh LB medium and incubated again about 1 h to cell Putative inner membrane protein Galactitol-1-phosphate dehydrogenase Putative malonate decarboxylase

R-CGGCTTTGCTGGCATTGTAG
Note: +, PCR product of expected size; À, no PCR product; d, differs among strains.I-Salmonella enterica, II-Salmonella.salamae, IIIa-Salmonella arizonae, density equal to 0.5 in McFarland standard.Cells were centrifuged (4000 rpm, 30 min, 4 C) and suspended in 3 mL of physiological saline and then 1 mL of this was transferred to 5 mL of fresh physiological saline and mixed with NHS in one-to-one proportion.Each strain was incubated with 50% NHS in a water bath (37 C, 180 min).Samples were collected at the beginning of the incubation (0 min, T 0 ) and after incubation (180 min, T 3 ), diluted (10 1 -10 6 ) and distributed (100 μL) on a solid nutrient agar medium (Biomaxima, Poland) by glass cell spreader.Plates were incubated overnight (37 C).The average number of Salmonella spp.colony-forming units (CFU/mL) was estimated from three tests.To confirm the NHS bactericidal activity, the assay was performed with decomplemented (inactivated by heating, 56 C, 30 min) inactivated human serum (IHS) as the control.

Colonial and morphological characteristics of Salmonella spp. isolates
Including all 30 isolates from studied reptiles (n = 30/69), we observed bacterial growth on different standard selective microbial media.On Simmons citrate agar, all isolates show blue colonies, indicating their ability to assimilate citrate as the only carbon source.Furthermore, they appear yellow-red on XLD agar, suggesting the ability to decarboxylate lysine and ferment xylose.Therefore, regarding the following biochemical features, such as the ability to (1) assimilate citrate and (2) decarboxylate lysine and ferment xylose, all isolates suggest the initial observation of typical Salmonella spp.(Table S1).However, on ChromAgar medium, 26/30, 87% isolates show mauve colonies, indicating their ability to produce esterase catalysing the hydrolysis of organic acid's esters, which is the feature of clinical Salmonella spp.samples (Heithoff et al., 2008;Kim et al., 2023;Maddocks et al., 2002;Pao et al., 2005;Svanevik & Lunestad, 2015).From a total of 30 isolates, 4 of them seem to be biochemically atypical, potentially environmental Salmonella spp.isolates, 13%-NN 12.2, II NN 6.1, 4.1S and II 4.1S.At the same time, 18/30 isolates appeared as nonpigmented, rounded colonies on MacConkey media, resembling Salmonella spp.and sharing biochemical characteristics similar to clinical samples of Salmonella spp.The other 12 isolates produce pink colonies (NN 1.1, NN 1.2, NN 1.3, NN 8.1, NN 9.2, NN 11.1, NN 14.3, III NN 14.3, III NN 14.5, III NN 14.6, 28.1S and 3.1L), suggesting the ability to utilise lactose.Furthermore, we noticed transparent colonies with black centres of 20 isolates (20/30, 67%) on SS medium (H 2 S producer) suggesting the growth of Salmonella sp.Thus, the other 10 isolates (10/30, 33%-NN 1.1, NN 1.2, II NN 4.7, III NN 14.3, 4.1S, 7.1S, 11S, 13.3S, II 16.2K and II 4.1S) do not produce H 2 S, what is atypical feature for clinical Salmonella spp.strains (Table 3).The identification of all (typical-consistent with clinical samples and atypical-inconsistent with clinical samples) Salmonella spp.isolated from N. natrix (n = 27) and N. fasciata (n = 3) is shown in Table S1.
Biochemical and serological identification of Salmonella spp.isolates On the API 20E test, all suspected Salmonella spp.isolates (n = 30) ferment glucose and arabinose, decarboxylate lysine and ornithine, assimilate citrate, and produce H 2 S and NO 2 (Table S1), which is consistent with clinical reference Salmonella sp.Furthermore, all Salmonella spp.are incapable of producing urea, do not break down tryptophan to indole and are devoid of cytochrome oxidase-the final enzyme of the respiratory chain in oxygen respiration (Table S1) exhibiting the same biochemical features as positive control sample: clinical reference Salmonella spp.In contrast, 77% of Salmonella spp.strains (23/30) synthesise βgalactosidase.
All Salmonella isolates were agglutinated with the polyvalent flagellar HM antiserum.In opposite to HM antiserum, other reactions with O group antisera and H: g,m flagella antiserum were negative.

Genetic identification of Salmonella spp. isolates
Based on the data from a multiplex-PCR assay with six primer pairs (STM4057, stn, invA, gatD, mdcA and fljB

ENVIRONMENTAL MICROBIOLOGY REPORTS
T A B L E 3 The juxtaposition of all experiments from this study allowing the identification of atypical Salmonella spp exhibiting different biochemical features than clinical reference Salmonella spp.strains.All samples isolated from Natrix natrix (n = 27) and Nerodia fasciata (n = 3) were analysed to identify Salmonella spp.The identification was performed by the observation of grown colonies in selective media, followed by API 20E tests and then, MALDI-TOF MS.All isolates prove the identification of Salmonella spp.Depending on which analysis is taken into account, we can detect different percentage of atypical Salmonella spp.strains.Note all samples should be considered to control samples, which are Salmonella reference strains isolated from human feaces.For the identification of all Salmonella spp.we refer the reader to Table S1.genes), we classified Salmonella isolates into different subspecies.Twenty-six isolates were characterised by the detection of stn, invA, mdcA and fljB genes as S. enterica subsp.diarizonae (Figure 1).In contrast, the remaining four isolates (4.1S, II 4.1S, II NN6.1 and NN 12.2) were demonstrated to be S. enterica subsp.houtenae because of the detection of only two of the stn and invA genes.The STM4057 and gatD genes were not detected in any of the tested samples.Table 2 represents the sizes, sequence primers and functions

Isolate
The classification of Salmonella isolates into different subspecies based on the detection of specific genes for each subspecies (according to Lee et al., 2009).In blue boxes we highlighted Salmonella spp.isolates identified as Salmonella enterica subsp.houtenae (4.1S, II4.1S, IINN.6.1 and NN12.2);M-DNA molecular weight marker.
The determination of genetic correlation of Salmonella spp.strains isolated from reptiles based on ERIC-PCR.(A) Agarose gel as in Figure 1 indicating strictly conserved fragments of the genome of studied Salmonella spp.isolates enabling the determination of the genetic similarities between them.(B) Cluster analysis of ERIC-PCR profiles of Salmonella spp. with their corresponding origin.The phylogenetic tree was generated by the average linkage agglomeration method (unweighted pair group method with arithmetic mean) using BioNumerics 7.6.2(Applied Maths).
of each gene, allowing us to differentiate Salmonella sp.isolates.

The determination of phylogenetic relationships-ERIC-PCR
Based on the ERIC-PCR reaction, all Salmonella spp.isolates from reptiles (n = 30) are genetically comparable.The cluster similarity cut-off value was set at least 69% (Figure 2).The overall Simpson's index of diversity for ERIC-PCR clustering was 0.69.However, the previous experiment indicated the detection of four Salmonella spp.isolates belonging to S. enterica subsp.houtenae (4.1S, II 4.1S, NN12.2 and II NN 6.1), we confirm that these strains differ genetically from the remaining tested isolates.Therefore, according to the phylogenetic tree presented in Figure 2, we generated two main clusters, one of which belongs only to S. enterica subsp.houtenae.Comparing these isolates, the genetic similarity was referred to as approximately 80%.The second cluster included all 26 isolates of S. enterica subsp.diarizonae.We classified them into two smaller subclusters, one of which consisted of three genetically similar isolates: 39.1K, 11S and 7.1S.These three strains were distinguished from the others by the lack of one of the two products weighing over 800 bp.The second subcluster was more diverse.However, even in this case, we were able to find some strong correlations between the genetic profiles of analysed Salmonella spp.isolates.For instance, according to the phylogenetic tree, isolates: NN 11.1 and NN 1.1 have substantial genetic similarities (approximately 97%).It is worth noting the profiles of Salmonella spp.isolates from the same snake species: N. fasciata (NF 9.2, NF 9.4 and NF 9.5).Their relatively close localization in the phylogenetic tree (with an additional product around 700 bp) and belongingness in the same subcluster reflect their high genetic similarity.This result also confirms comparable proteomic and biochemical characteristics observed in previously described experiments (culture methods, API 20E test and MALDI-TOF MS, Table 3).

Virulence genotyping
All tested Salmonella spp.were genotyped by PCR as described (Skyberg et al., 2006).The presence of 12 out of 17 amplified genes was confirmed.The VG were categorised into three distinct virulotypes (VT): A, B and C. The full gene was the only difference compared to the A type.All of the S. houtenae strains had identical VT (C) and the PCR confirmed the presence of prgH and sifA gene only in this VT.All the tested Salmonella spp.strains contained msgA, tolC, sitC, sipB and sopB genes.VT A and B contained additional invA, orgA and spiA genes and were the most common (VT-A in 67% and VT-B in 20% of tested strains).The only difference between VT-A and VT-B was the ironN gene, presented in VT-B and VT-C, but not in VT-A type.All of the strains with VT-A and VT-B were classified as S. enterica subsp.diarizonae.SifA and prgH genes were detected only in VT-C, which was presented in each (4/4) S. enterica subsp.houtenae strains; these genes were not detected in other isolates.None of the strains contained the spvB, pagC, lpfC, pefA and spaN genes (Table 4).

Normal human serum activity
As mentioned before, selected Salmonella spp.strains isolated from N. natrix (n = 5) and N. fasciata (n = 3) were tested for bactericidal action of NHS.At the beginning of incubation with NHS, the average cell count ranged from 2.5 Â 10 6 to 4.9 Â 10 6 CFU/mL.All of the tested Salmonella spp.strains isolated from N. natrix were resistant to bactericidal action of 50% NHS, whereas all the strains isolated from N. fasciata were susceptible.Two strains (NN 1.3 and 28.1K) demonstrated a highly resistant level to bactericidal action of NHS and obtained 1.0 Â 10 8 and 4.3 Â 10 7 CFU/mL, respectively (Table 5).The remaining of the tested strains isolated from N. natrix (II 4.1S, NN 13.3 and 39.1K) demonstrated lower but still serum resistance to NHS and reached 608%, 615% and 358% of survival, respectively, after 3 h of incubation.The count of the colonies of strains isolated from N. fasciata (NF 9.2, NF 9.4 and NF 9.5) did not exceed the 4.8 Â 10 3 CFU/mL after 3 h of incubation with NHS, which was less than 1% of survival.All of the strains isolated from both N. natrix and N. fasciata survived after 3 h control incubation with ISH and reached cell count 2.5 Â 10 7 to 1.6 Â 10 8 CFU/mL (Table 5).The most serum-resistant strains (NN 1.3 and 28.1K) also demonstrated the best growth in control conditions.Strains susceptible to the NHS were able to grow with IHS.

Antimicrobial susceptibility
All the Salmonella spp.strains were susceptible to 13 of the14 antimicrobial agents used in this study and 80% of strains were susceptible to all tested antibiotics.Growth inhibition zones with antibiotics are presented in Table 6.Tigecycline was the only ineffective antimicrobial agent for some of the tested Salmonella spp.strains.All the strains resistant to tigecycline were S. enterica subsp.diarizonae isolated both from N. natrix (7.1S, 13.3S, NN 1.1, NN 1.2 and III NN 14.5) and N. fasciata (NF 9.5).

T A B L E 4
The taxonomic classification of all Salmonella spp.isolated from analysed reptiles (based on data from Lee et al., 2009) and the determination of their virulotypes (according to Skyberg et al., 2006).Note virulotypes A and B belong to Salmonella diarizonae, while virulotype C belongs to Salmonella houtenae.

Salmonella spp. prevalence in free-living snakes
Our study concerning the prevalence of Salmonella spp. in reptiles is the first on such a large sample of free-living snakes in Europe.We indicated that freeliving N. natrix are common carriers of Salmonella spp.This finding gives us strong evidence that Salmonella spp.rods are a part of the intestinal microbiota of snakes native to Poland.Another study on N. natrix in Poland but on a smaller number of snakes (n = 15) showed that 87.5% of them were Salmonella spp.carriers (Zając et al., 2016).The finding of Salmonella sp.prevalence in reptiles was also shown in our previously reported studies.Hacioglu et al., 2015).In contrast, studies from Germany showed that Salmonella spp. was not isolated from the intestinal tracts of free-living N. natrix (n = 12).The same study showed that 8/23 (34.8%) free-living Vipera berus were the carriers of Salmonella spp.(Schmidt et al., 2014).Furthermore, Luka c et al. found no Salmonella spp.isolates in the gastrointestinal tracts of free-living four-lined snakes Elaphe quatuorlineata (n = 20) (Lukač et al., 2017).Those limited European studies on free-living snakes show opposite results, which may indicate a high population-or/and species-specificity.A greater number of populations and species are needed to be scanned for the presence of Salmonella spp. to assess environmental correlates of Salmonella prevalence.
To date, the highest prevalence of Salmonella spp. was recorded in Europe in captive reptiles (Bjelland et al., 2020;Bošnjak et al., 2016;Dudek et al., 2019;Hydeskov et al., 2013;Marin et al., 2021;Pees et al., 2013;Russo et al., 2018;Wikström et al., 2014).Our study populations are located within a large city and in its close vicinity, which is associated with a high risk of contact between wild snakes and captive ones that are accidentally or deliberately released into nature.This phenomenon raises the question of whether Salmonella spp.occurring in free-living versus captive populations are independent strains or if the frequent occurrence of Salmonella spp. in wild snakes can originate through contact with other animals naturally existing in the environment rather than captive snakes.For instance, Bauwens et al. reported the presence of Salmonella spp.rods outside the terrariums of captive reptiles, which indicates that the pathogenic bacteria can be easily transmitted to the environment by domestic animals and/or humans (Bauwens et al., 2006).This indicates that Salmonella can probably be easily transferred from captive reptiles to the environment via the activity of humans.In our study, grass snakes were collected in Crac ow and closely located sites.These areas are densely populated with a well-developed herpetoculture and cases of released into the nature of alien reptiles are regularly reported.This results in an increase in the risk of contact with native species, which-together with easiness of Salmonella spread-raises the question of the role of captive reptiles in the distribution of Salmonella in the wild (Morydz et al., 2017).Reptiles can be colonised by Salmonella spp. at the very early stage of life through transovarial transmission, direct contact with other reptiles or with their faeces (Mermin et al., 1997).This suggests the maintenance of microbiota through generations, which increases the time window over which Salmonella can be transferred into the environment.
Thus, preventing the zoonotic risk associated with Salmonella spp.occurring in free-living snakes should be focused on controlling captive reptiles' maintenance.In addition, an essential aspect of zoonotic control can be related to the conservation of natural populations of reptiles.This is because harvesting or deliberate persecution of free-living species is associated with direct contact with animals, which is a risk factor for infection.A well-known example of snake harvesting is in Asian wet markets, including Wuhan in China, where the COVID-19 pandemic originated.Secondly, greater densities of the host population can reduce the prevalence of particular parasites of pathogens due to the dilution effect and vice versa-a vastly decreased population number can be predicted by the locally elevated density of several pathogens and increase the risk of their further spread, for example, into humans.
There are many other anthropogenic factors increasing zoonoses spreading: poverty and culinary traditions of wild animals' meat consumption, climate change resulting in the emergence of new species in places where they did not exist before, urbanisation of the natural environment of wild animals' occurrence, an increase in the frequency and distance of human travel and international trade (Magouras et al., 2020).

Virulence
Invasive NTS and serious NTS in patients from risk groups require antibiotic treatment, which efficiency can be reduced by drug resistance of particular T A B L E 5 Colony-forming units (CFU) per millilitre and percent of survival of Salmonella spp.cells after 3 h incubation in 50% normal human serum (NHS) and 50% inactivated human serum (IHS).et al., 2022;Nishio et al., 2005;Ondari et al., 2019;Rossi et al., 2019).In Europe, Poland has one of the highest levels of drug-resistant Salmonella spp.strains, including multidrug-resistant strains (MDR) and extended-spectrum beta-lactamases (ESBL) strains (EFSA & ECDC, 2019).In most cases, Salmonella spp.strains found in reptiles reveal mild to strong levels of drug resistance.In our study, we reported mildly potent drug resistance to only tigecycline in five serovars isolated from free-living N. natrix and one strain isolated from captive N. fasciata.Tigecycline is an important drug active against many bacteria, including drugresistant pathogens like methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE), vancomycinresistant Enterococcus species (VRE), extended-spectrum-beta-lactamases-producing Enterobacteriaceae and multi-drug and extensively-drug-resistant (MDR) Acinetobacter baumannii (Kim et al., 2016;Townsend et al., 2007).Tigecycline is a recommended drug for the treatment of serious bacterial infections, like complicated skin and tissue infections, intra-abdominal infections, pneumonia and osteomyelitis and is currently the last-line drug against MDR bacterial pathogens, including carbapenem-resistant Enterobacteriaceae (Yaghoubi et al., 2022).Tigecycline-resistant Salmonella spp. is rarely described; however, high percentage (93.1%) of non-susceptible (intermediate or resistant) Salmonella isolated from healthy reptiles have been reported in Italy (Bertelloni et al., 2016).Tigecycline-resistant Salmonella spp.were also isolated from poultry production areas and humans (Wang et al., 2023, Abd El-Aziz et al., 2021).It has been described that the activity of AcrAB-TolC efflux pump system and the presence of tigecycline-resistant gene tet(X4) are two main mechanisms of resistance to this drug (Chetri et al., 2019;Zhang et al., 2022).We found the tolC gene in each Salmonella virulotype described in this research and further analyses will be performed.Other studies on European snakes, for example, Schmidt et al. reported resistance to streptomycin of Salmonella spp.strains isolated from free-living Vipera berus (Schmidt et al., 2014).Research by Cota et al., similar to ours, indicates low resistance of Salmonella spp.isolates from reptiles to antibiotics.Salmonella spp.and Salmonella IIIb obtained from Pantherophis guttatus guttatus and Python regius were sensitive to tested fluoroquinolones, aminoglycosides, amoxicillin with clavulanic acid, and ampicillin but resistant to penicillin (Cota et al., 2021).On the other hand, Salmonella Kentucky isolated from snakes in Poland were resistant, among other drugs, to ampicillin and ciprofloxacin (Zając et al., 2013).It is crucial for RAS-causing Salmonella to be treatable, as seen with Salmonella IIIb isolated in Romania, which was sensitive to ampicillin, ciprofloxacin, trimethoprim-sulfamethoxazole, and third-generation cephalosporin (Gavrilovici et al., 2017).However, in Romania, there was already isolated resistant Salmonella spp., including resistance to first-and second-generation cephalosporins and aminoglycosides.(Cristina et al., 2022).Marin et al. (2021) studied reptiles from households and pet shops, where 72% of strains were MDR, and most frequently they observed gentamicin-colistin and gentamicin-colistin-ampicillin resistance patterns.MDR strains were also reported in Wuhan, China-the epicentre of the COVID-19 outbreak.In 2020, Xia Y et al. isolated MDR Salmonella spp.from the lungs of edible snakes with pneumonia.The strain was resistant to 14 antibiotics.Moreover, the authors conducted the lethal test of isolated Salmonella spp.serovar in chickens, resulting in 75% mortality in chickens in 24 h (Xia et al., 2020).This example strongly underlines the zoonotic potential of Salmonella spp.strains isolated from snakes and its presence in food chains and the possibility of infecting people.
Besides the drug resistance, we tested the bactericidal action of 50% NHS on isolated Salmonella spp.strains.All strains isolated from free-living N. natrix were resistant, and the strains isolated from captive N. fasciata were sensitive to NHS action.Given that the immunity of infants and small children is highly based on innate immune response mechanisms such as complement contained in human serum, the resistance of bacteria to NHS can be one of the factors leading to invasive infections.In our previous studies, we indicated that Salmonella strains even from the same serotype group vary in their susceptibility to NHS.Many strains were sensitive to the bactericidal action of serum.Multiple passages of some strains in serum led to achieving resistance to NHS (Bugla-Płosko nska et al., 2009;Pawlak et al., 2017).In the present study, all tested strains were resistant to human serum, which suggests that the long-term carrier of Salmonella strains in reptile intestines might be a factor that leads to serum resistance.
We analysed the presence of genes that are the main determinants of virulence in vertebrates (Alix et al., 2008;Dudek et al., 2019;Gunn et al., 1995;Krawiec et al., 2015;Lilic et al., 2010;Skyberg et al., 2006;Stein et al., 1996).We detected 12 of the 17 amplified genes in tested Salmonella spp.strains.However, essential bacterial invasion VG are missing, including spvB, pagC, lpfC, pefA and spaN.These genes are responsible for toxicity, cell adhesion, survival within macrophages, export and assembly of fimbrial subunits, and entry into host cells, respectively.Pasmans et al. (2005) found pef A only in one (from 79) Salmonella spp.strain isolated from captive reptiles.Bertelloni et al. (2016) detected spv genes (important for macrophage cytotoxicity and destabilisation of the cytoskeleton of the eukaryotic cell) also in only one (from 29) Salmonella spp.serovar.In our previous study, Dudek et al., aimed to determine VGs profiles for both S. enteritidis from humans and Salmonella strains from reptiles housed in a Polish zoo (Dudek et al., 2019).Salmonella strains from reptiles revealed lower prevalence of VG compared to Salmonella strains isolated from humans.In line with our study, the sitC gene was observed in all tested Salmonella strains.However, genes that were not detected in our Salmonella-tested strains (Table 4) were highly expressed in Salmonella strains isolated from captive reptiles, including, lpfC (27%), spaN (47%) and pagC (80%) genes (Dudek et al., 2019).Furthermore, 93% of clinical Salmonella strains possessed the invA gene; its expression was also observed among our tested environmental strains, except those identified as S. houtenae (4.1S, II 4.1S, NN12.2 and II NN 6.1).Additionally, as Dudek et al., determined, gene cdtD was not present in clinical Salmonella strains, it was expressed in 40% of reptilian Salmonella strains.In our case, this gene was present in all Salmonella strains, except those belonging to S. houtenae.In line with Dudek et al. (2019) study, none of the tested Salmonella strains isolated from reptiles had spvB and pefA genes in its genome.MDR Salmonella spp.strain isolated by Xia et al. (2020) despite its high mortality in chickens and ability to cause pneumonia in snakes, also lacks spv genes.In this research, we classified the isolated strains into two subspecies: S. enterica subsp.diarizonae and S. enterica subsp.houtenae.The strains of the subspecies diarizonae showed phenotypic and genotypic variability, which may be the basis for further research, including the typification of surface antigens.

Final remarks
Our study indicates that free-living snakes in Europe can be carriers of Salmonella spp.strains that have poor ability to invade host cells; however, with resistance to human serum.The bacteria should be treated with clinical importance to risk groups, especially infants and very young children.So far, to our knowledge, it is the biggest study on snakes living in Europe.It is still not fully clear whether Salmonella spp. is a part of the natural intestinal microbiota of reptiles or an opportunistic pathogen.Most reptiles do not show any symptoms of salmonellosis; however, some factors including stress, diet change, food deprivation, exposure to cold, or transportation may develop symptoms such as diarrhoea, vomiting, anorexia, pneumonia, and sepsis, leading even to death (CDC, 2006;Ebani et al., 2005;Bjelland et al., 2020;Xia et al., 2020).It is a very interesting phenomenon, as for people Salmonella spp. is a virulent pathogen and transmission of Salmonella spp.serovars from reptiles to humans can lead to RAS.RAS cases are not monitored in Poland and are poorly monitored in some European countries, for example, Germany (Pees et al., 2013;Schneider et al., 2009), Belgium (Meervenne et al., 2009), France (Perez Costa et al., 2015;Horvath et al., 2016), the Netherlands (Mughini-Gras et al., 2016), Norway (Bjelland et al., 2020), Portugal (Cota et al., 2021), Romania (Gavrilovici et al., 2017), and Spain (Bertrand et al., 2008;Perez Costa et al., 2015;Harris et al., 2010).On the other hand, the United States struggles with RAS and monitors the cases, noting even local outbreaks (Cain et al., 2009, Meyer Sauteur et al., 2013, Whiley et al., 2017, Centers for Disease Control and Prevention [CDC], 2020).RAS is mainly a health problem for young children, immunocompromised patients, and the elderly population.The younger the child is, the more considerable the risk of invasive salmonellosis.Although turtles are reported to be the most often source of Salmonella rods causing children's RAS, the invasive forms of Salmonella are transmitted more often from other reptiles, including lizards and snakes (Vora et al., 2012).We indicated the occurrence of VG (invA, sipB, prgH, orgA, tolC, iroN, sitC, sifA, sopB, spiA, cdtB and msgA) in three distinct VT.Different VG are parts of pathogenicity islands in Salmonella spp.serovars and can be widely distributed among strains isolated from many animal species (Krawiec et al., 2015;Mokracka et al., 2018).Furthermore, based on the growth on standard media (especially, ChromAgar, MacConkey and SS media), we determined most of the tested reptilian Salmonella strains are biochemically atypical compared to clinical strains-their growth looks different compared to clinical samples (Tables 4 and S1).Based on the API20E Test, we identified β-galactosidase synthesised by 23 Salmonella spp.serovars.This is a poorly known aspect of Salmonella spp.biology, which can be serovar-and pathovar-specific (Nuccio & Bäumler, 2014), and a result of adaptation to a new host or environment.There is a relevant difference in biochemical properties of gastrointestinal and extraintestinal Salmonella spp.serovars.The presence of βgalactosidase in Salmonella spp. is often detected in S. arizonae and S. diarizonae.These are the so-called late fermenting subspecies (Lamas et al., 2018;Lenev et al., 2016).Based on the literature, this biochemical property is not connected with the higher virulence of serovars.The role of β-galactosidase, its variation for Salmonella spp.strains and association with environmental factors need further investigation.
-arrangement No. 2039/12/2018 and No. annexe 0242/02/2020.The serum material was collected under the applicable rules included in the Act on the Public Blood Service of May 20, 2016, and in Directive 2002/980/EC of the European Parliament and the Council of 27 January 2003.
T A B L E 1 Oligonucleotide primers and expected band patterns of each Salmonella species or subspecies.
Abbreviation: VT, virulotype.a Each Salmonella spp.isolate was categorized to different type of virulence (A-in orange, B-in blue, C-in green).
with the consent of the Local Ethical Committee-resolution no.132/2015 of 26 May 2015 and resolution No. 73/2017 of 16 February 2017.